Standard techniques used for the immunologic monitoring of cancer vaccine trials have included tritiated thymidine incorporation as a measure of antigen specific proliferation and the ability of cytotoxic T cells (CTL ) to lyse a tumor, most often assessed by chromium release. These two methods are difficult to reliably produce, require in vitro expansion, and are not quantitative. Addition of these assay systems to a limiting dilution analysis format has allowed a more accurate assessment of precursor frequency, but the number of immune effector cells needed to establish the analysis requires patients undergo a leukapheresis. Thus, these strategies are not viable for wide-scale immunologic monitoring. Antigen specific immune responses are largely regulated by secretion of cytokines whose function is to govern the growth and differentiation of T cell populations. Secretion of cytokines by T cells responding to a specific antigen has become a common measure of a functional immune response. Although the measurement of cytokine production by T cells using ELISA methodologies is not quantitative, measurement of cytokines offers a detailed characterization of the function of the T cell and the phenotype of the immune environment generated after antigen recognition. Recently, a single-cell assay has been developed to more accurately assess cytokine-producing cells. The method, termed ELIspot (enzyme-linked immunosorbant spot) can detect antigen reactive T cells before such cells could produce sufficient amount of protein to be detected by ELISA. Although ELIspot has become the "comparator" assay for immunologic monitoring, the sensitivity and limits of detection of the assay vary greatly from laboratory to laboratory . Obstacles to be overcome to translate ELIspot from a laboratory tool to a clinical grade monitoring technique include maximizing assay parameters to avoid any in vitro expansion step, develop the assay for use in cryopreserved cells, determine optimal antigen preparations used in analysis, and define the reliability of the assay to perform over time on the in multiple clinical samples. The specific aims of this proposal are to: (1) determine whether ELIspot can identify the T cell response to foreign protein antigens from the PBL of volunteer donors and cancer patients, (2) determine whether ELIspot can identify the T cell response to tumor antigens from the PBL of volunteer donors and cancer patients, (3) determine whether the T cell response, as measured by ELIspot, predicts tumor protection in murine transgenic tumor models such as the CEA and neu transgenic mice, and, finally, (4) determine whether ELIspot can predict immunization to cancer antigens and whether levels achieved are that similar to infectious disease antigens in human clinical trials of CEA, HER2, gp 100, and MAGE3 vaccines.